Cationic membrane proteins are surprisingly common in nature and carry out a wide variety of functions such as immune defense, drug delivery, and ion channel gating. Yet how cationic residues, especially Arg, overcome the free-energy barrier to insert into the hydrophobic part of the lipid membrane is poorly understood. Structure determination of cationic membrane proteins has lagged behind that of hydrophobic membrane proteins, thus limiting our understanding of many health-related cellular processes such as innate immunity and ion channel gating. The broad, long-term objective of this work is to elucidate the structural basis for the insertion and translocation of cationic proteins across lipid membranes. The general approach is to determine the atomic-level structure and the membrane topology depth of insertion and orientation of cationic membrane proteins using high-resolution solid-state NMR spectroscopy. The ability to probe structural information directly in the lipid membrane and the site specificity afforded by high-resolution magic-angle spinning NMR are unique advantages of our structure determination approach. We propose to investigate two antimicrobial peptides of mammalian origin and a cell-penetratin peptide, all rich in Arg. Based on our finding in the last grant period; we hypothesize that guanidinium cations insert into the hydrophobic part of the lipid membrane by complexing with the lipid phosphate anions, in so doing creating either transient or permanent membrane defects. We will test this hypothesis by 1) a comparative study of the structure and lipid-interaction of Arg-removed, Arg-altered, and Arg- dimethylated variants of the 2-sheet antimicrobial peptide PG-1, to assess the relative importance of charge-charge attraction versus hydrogen bonding to guanidinium-phosphate complexation and to the eventual membrane-disruptive activity of PG-1; 2) determining the complete three-dimensional structure of a human antimicrobial peptide, human 1-defensin-1, in the membrane, elucidating its depth of insertion and orientation, and identifying the location of Arg residues in the membrane; 3) investigating the structure and mechanism of action of a cell-penetrating peptide, penetratin, to compare with the mechanisms of antimicrobial peptides we studied before. This research will employ a wide variety of advanced solid-state NMR techniques, such as multidimensional correlation techniques, 1H and 19F spin diffusion, internuclear distance measurements, paramagnetic relaxation enhancement, and 31P lineshapes of macroscopically aligned membranes. The resulting structural information will help the design of more potent antibiotics to combat bacterial resistance and better drug-delivery compounds to cross cell membranes. PUBLIC HEALTH RELEVANCE: The proposed research has broad health relevance in two areas. It will provide a better structural basis for designing new antimicrobial agents to serve as potent antibiotics without resistance. It will also benefit the development of more effective drug-delivery compounds that cross the cell membrane without damaging its integrity. [unreadable] [unreadable] [unreadable]